Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include features (sometimes referenced as spots or regions) of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. The array is “addressable” in that different features have different predetermined locations (“addresses”) on a substrate carrying the array.
Biopolymer arrays can be fabricated using in situ synthesis methods or deposition of the previously obtained biopolymers. The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA). In situ methods also include photolithographic techniques such as described, for example, in WO 91/07087, WO 92/10587, WO 92/10588, and U.S. Pat. No. 5,143,854. The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different feature locations on the substrate to yield the completed array. Washing or other additional steps may also be used. Procedures known in the art for deposition of polynucleotides, particularly DNA such as whole oligomers or cDNA, are described, for example, in U.S. Pat. No. 5,807,522 (touching drop dispensers to a substrate), and in PCT publications WO 95/25116 and WO 98/41531, and elsewhere (use of an ink jet type head to fire drops onto the substrate).
In array fabrication, the quantities of DNA available for the array are usually very small and expensive. Sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require the manufacture and use of arrays with large numbers of very small, closely spaced features.
The arrays, when exposed to a sample, will exhibit a binding pattern. The array can be interrogated by observing this binding pattern by, for example, labeling all polynucleotide targets (for example, DNA) in the sample with a suitable label (such as a fluorescent compound), scanning an interrogating light across the array and accurately observing the fluorescent signal from the different features of the array. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample. Peptide arrays can be used in a similar manner. Techniques for scanning arrays are described, for example, in U.S. Pat. Nos. 5,763,870 and 5,945,679. However, the signals detected from respective features emitted in response to the interrogating light, may be other than fluorescence from a fluorescent label. For example, the signals may be fluorescence polarization, reflectance, or scattering, as described in U.S. Pat. No. 5,721,435.
Array scanners typically use a laser as an interrogating light source, which is scanned over the array features. Particularly in array scanners used for DNA sequencing or gene expression studies, a detector (typically a fluorescence detector) with a very high light sensitivity is normally desirable to achieve maximum signal-to-noise in detecting hybridized molecules. At present, photomultiplier tubes (“PMTs”) are still the detector of choice although charge coupled devices (“CCDs”) can also be used. PMTs are typically used for temporally sequential scanning of array features, while CCDs permit scanning many features in parallel.
Laser output power in such array scanners may tend to drift over time. As described in U.S. Pat. No. 5,763,870, it is known to provide an integral power regulation sensor for a laser which is used to monitor laser output power. The power sensors (that is, laser light illuminance sensors) are connected to current-regulating circuitry that varies the supply current to the laser and responds to changes in output power. For gas lasers, the power sensors may be mounted internally and a beam splitter redirects a portion of the output beam energy to the power sensor, which may be a photodiode. For semiconductor lasers, the power sensor may be formed on the same substrate as the semiconductor layers that define the laser device, such as described in U.S. Pat. Nos. 5,323,026 and 4,577,320. However, the present invention recognizes that for some types of gas lasers it may be difficult to reliably switch between two power levels quickly, and that for diode lasers changing the laser power may have the undesired side effect of changing the wavelength.
The present invention further realizes that strong signals may occur in response to the interrogating light, either from bright features or from other components (for example, fluorescence of the glue holding an array substrate in a housing). In PMTs and other detectors (such as CCDs) very strong signals that are (depending on the type of detector) either spatially and/or temporally close to weak signals, may undesirably affect the latter. For example, a PMT reading a very bright signal at a given time, may change its sensitivity for a short time after this, or on a CCD detector very bright pixels may bleed their charge into adjacent ones. Extremely strong signals may even damage either kind of (normally expensive) detector. In any event, the accurate detection of signals from an array being interrogated by the scanner may be in doubt due to such effects.
The present invention realizes that it would be desirable then, to provide a technique for scanning an addressable array which allowed for rapid correction in variations in power of an interrogating light. The present invention further realizes that it would be desirable if some means were provided during array scanning, to limit exposure of a detector to very strong signals generated by features or other sites in response to the interrogating light. Additionally, the present invention also realizes that during the typically rapid scanning of an array, some types of light sources may not respond sufficiently rapidly to changes in power input (as discussed above) to allow for the corrections of the present invention while maintaining high array scanning speeds.